Part:BBa_K2238003

Glucose oxidase (A. niger) Quadruple Mutant, Cysteine tagged

Coding sequence for the Aspergillus niger glucose oxidase enzyme which is a reductase that catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide. This part contains the E. coli codon optimized sequence for the A. niger wild type glucose oxidase. It requires the cofactor, flavin adenine dinucleotide (FAD). FAD acts as the initial electron acceptor, in a redox reaction, and is reduced to FADH2. Then FADH2 is oxidized by molecular oxygen, which is then reduced to hydrogen peroxide.

This mutant is reported to have higher stability as compared to wild type and has been modified with a 3X-cysteine C-terminal.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 36
  Illegal BamHI site found at 346
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 370
  Illegal BsaI.rc site found at 1588
  

Functional Parameters

GOx catalyzes the oxidation of D-glucose and produces hydrogen peroxide (H₂O₂). We took advantage of the Invitrogen Molecular Probes Amplex® Red Glucose/Glucose Oxidase Assay Kit (A22189) which allows for a one-step detection of glucose oxidase activity by coupling the production of H₂O₂ to the activity of horseradish peroxidase (HRP). H₂O₂ reacts with Amplex® Red (a colorless molecule), in the presence of HRP, to yield resorufin (red-fluorescent product). This colorimetric assay directly couples the activity of glucose oxidase to the production of resorufin.

Three of our GOx mutants were expressed and then purified by nickel resin. Each purification was eluted in 5, 1 ml elutions (phosphate buffer, 250 mM imidazole, pH 7.0). All the variants were expressed under the same conditions and lysates were prepared by addition of lyticase, followed by sonification (in phosphate buffer, pH 7.0). The negative elution control corresponds to the expression of an empty plasmid carrying no GOx variant genes. This negative control was also prepared for the nickel column and eluted, as if HIS-tagged proteins were present.

The figure below depicts the results of our Amplex assay. In each well there was 50 µl of the reaction buffer which contains HRP, glucose, and Amplex® Red. We then added 50 µl of the assay kit GOx to the "+" control well, 50 µl of elution buffer only to the negative buffer control well, and 50 µl of each elution to the indicated wells. The reaction was incubated in the dark for 30 min at room temperature. By simple colorimetric analysis, there is obvious GOx activity in all of our GOx variants that we isolated. The GOx-4mut-cys lane corresponds to the activity of this biobrick (K2238003). There was GOx activity detected in elutions 2,3 and 5 for GOx-4mut-cys, however at a reduced level as compared to the control and our other GOx variants. Even though we observed a reduced amount of activity for these samples this could be due to several variables that are still being examined. These results do conlcude that this brick works as expected because it oxidizes glucose to produce H₂O₂.

GOx-Wt (part BBa_K2238000); GOx-4mut (part BBa_K2238001); GOx-4mut-cys (part BBa_K22380003)

We are currently working toward quantifying its activity in comparison to a standard of pure Wt glucose oxidase.